PROJECT SUMMARY Legionella pneumophila (Lp) is the agent of Legionnaires' disease, a common and potentially fatal form of pneumonia. Humans come in contact with Lp by inhaling contaminated water droplets produced by aerosol- generating devices. Lp flourishes in water systems by growing in a wide variety of amoebae. Thus, deciphering how Lp infects amoebae is critical to our understanding of the transmission of disease. Recently, we found that the cytoplasmic Cas2 protein of Lp promotes bacterial infection of multiple amoebae; e.g., cas2 mutants, but not their complemented derivatives, exhibit a 1000-fold defect in Acanthamoeba castellanii. The Cas2 family of proteins is well known for its role in the bacterial and archeal CRISPR-Cas system that constitutes a form of adaptive immunity against phage and plasmid. However, the infection event mediated by Lp Cas2 is distinct from this function, because cas2 mutants exhibit their infection defects in the absence of phage or plasmid and mutants lacking the CRISPR array or any one of the other cas genes are not impaired in infection. Our most recent studies further show that purified Cas2 of Lp has RNase and DNase activity, with the RNase activity being most pronounced. By characterizing a catalytically deficient version of Cas2, we determined that nuclease activity is critical for promoting infection of amoebae. Remarkably, introduction of Cas2, but not its catalytic mutant form, into a strain of Lp that naturally lacks a CRISPR-Cas locus caused that strain to be 80-fold more infective for amoebae, unequivocally showing that Cas2 facilitates the infection process independently of any other component encoded by the CRISPR-Cas locus. Overall, these data document that the acquisition and retention of the Cas2 nuclease provides a unique, selective advantage for Lp in environmental niches that are critical for the transmission of disease. Furthermore, they are a novel demonstration of a non-canonical role for Cas2. Thus, we now hypothesize that Lp Cas2, by virtue of its ability to cleave, process, or degrade RNA, modulates an mRNA or sRNA that encodes or regulates an infectivity determinant(s). We further posit that the specificity of this Cas2 activity is achieved in the context of the bacterial cell by the interaction between Cas2 and another (non-CRISPR-Cas) protein or proteins. The experiments proposed in this application aim to uncover the target(s) of the Cas2 RNase (Aim1) and identify interacting partners of Cas2 that might be helping to guide its target selection (Aim 2). Given that Cas2 is conserved across all CRISPR-Cas systems, it is likely that our findings concerning the Cas2 nuclease activity will have implications for many other bacteria and archaea, including many pathogens. In sum, this innovative proposal will i) elucidate a novel infectivity determinant, ii) continue to reshape our view of CRISPR-Cas as having roles outside of phage/plasmid immunity, and iii) uncover a new form of gene regulation.